Oxidase-Peroxidase Method of Ethanol Assay in Fermented Musts and Wine Products

A new alcohol oxidase-peroxidase method of ethanol assay in fermented musts and wine products is described and compared to conventional methods routinely used in winemaking. The sensitivity, accuracy, and reliability of this method were determined. The results of ethanol determination in fermented musts and wines correlated well with the data obtained by refractometry (correlation coefficient R = 0.9595, p < 0.0001) and densitometry (correlation coefficient R = 0.9384, p < 0.0001). The proposed method is less time- and labor-consuming and allows simultaneous analysis of a series of wine samples.     

Rapid screening of the fermentation profiles of wine yeasts by Fourier transform infrared spectroscopy

A rapid screening method for the evaluation of the major fermentation products of Saccharomyces wine yeasts was developed using Fourier transform infrared spectroscopy and principal component factor analysis. Calibration equations for the quantification of volatile acidity, glycerol, ethanol, reducing sugar and glucose concentrations in fermented Chenin blanc and synthetic musts were derived from the Fourier transform infrared spectra of small-scale fermentations. The accuracy of quantification of volatile acidity in both Chenin blanc and synthetic must was excellent, and the standard error of prediction was 0.07 g l(-1) and 0.08 g l(-1), respectively. The respective standard error of prediction in Chenin blanc and synthetic musts for ethanol was 0.32% v/v and 0.31% v/v, for glycerol was 0.38 g l(-1) and 0.32 g l(-1), for reducing sugar in Chenin blanc must was 0.56 g l(-1) and for glucose in synthetic must was 0.39 g l(-1). These values were in agreement with the accuracy obtained by the respective reference methods used for the quantification of the components. The screening method was applied to quantify the fermentation products of glycerol-overproducing hybrid yeasts and commercial wine yeasts. Principal component factor analysis of the fermentation data facilitated an overall comparison of the fermentation profiles (in terms of the components tested) of the strains. The potential of Fourier transform infrared spectroscopy as a tool to rapidly screen the fermentative properties of wine yeasts and to speed up the evaluation processes in the initial stages of yeast strain development programs is shown.     

Influence of the physiological state of the inoculum on fermentation of musts from Pedro Ximénez grapes by Saccharomyces cerevisiae

Two inocula in different physiological states, namely in the exponential growth phase and in the declining phase were prepared from a strain of Saccharomyces cerevisiae. With these inocula were fermented musts from grapes of the Pedro Ximénez variety, sterilized by filtration. Cell growth and the activity of the enzymes alcohol dehydrogenase and aldehyde dehydrogenase [NADP+ and NAD(P)+] were found to vary with the state of the inoculum. This was reflected in the specific rate of production and, in some instances, in the final concentration of acetaldehyde, acetic acid, ethanol, isoamyl alcohols, phenethyl alcohol and their esters in the wine.     

Amperometric biosensor for ethanol analysis in wines and grape must during wine fermentation

The amperometric biosensor for ethanol determination based on alcohol oxidase immobilised by the method of electrochemical polymerization has been developed. The industrial screen-printed platinum electrodes were used as transducers for creation of amperometric alcohol biosensor. Optimal conditions for electrochemical deposition of an active membrane with alcohol oxidase has been determined. Biosensors are characterised by good reproducibility and operational stability with minimal detection limit of ethanol 8 x 10(-5) M. The good correlation of results for ethanol detection in wine and during wine fermentation by using the developed amperometric biosensor with the data obtained by the standard methods was shown (r = 0.995).     

Reduction of volatile acidity of acidic wines by immobilized Saccharomyces cerevisiae cells

Excessive volatile acidity in wines is a major problem and is still prevalent because available solutions are nevertheless unsatisfactory, namely, blending the filter-sterilized acidic wine with other wines of lower volatile acidity or using reverse osmosis. We have previously explored the use of an empirical biological deacidification procedure to lower the acetic acid content of wines. This winemaker’s enological practice, which consists in refermentation associated with acetic acid consumption by yeasts, is performed by mixing the acidic wine with freshly crushed grapes, musts, or marc from a finished wine fermentation. We have shown that the commercial strain Saccharomyces cerevisiae S26 is able to decrease the volatile acidity of acidic wines with a volatile acidity higher than 1.44 g?L^?1 acetic acid, with no detrimental impact on wine aroma. In this study, we aimed to optimize the immobilization of S26 cells in alginate beads for the bioreduction of volatile acidity of acidic wines. We found that S26 cells immobilized in double-layer alginate–chitosan beads could reduce the volatile acidity of an acidic wine (1.1 g?L^?1 acetic acid, 12.5 % (v/v) ethanol, pH 3.12) by 28 and 62 % within 72 and 168 h, respectively, associated with a slight decrease in ethanol concentration (0.7 %). Similar volatile acidity removal efficiencies were obtained in medium with high glucose concentration (20 % w/v), indicating that this process may also be useful in the deacidification of grape musts. We, therefore, show that immobilized S. cerevisiae S26 cells in double-layer beads are an efficient alternative to improve the quality of wines with excessive volatile acidity.     

Technological properties of indigenous wine yeast strains isolated from wine production regions of Turkey

The purpose of this study was to evaluate the important technological and fermentative properties of wine yeast strains previously isolated from different wine producing regions of Turkey. The determination of the following important properties was made: growth at high temperatures; fermentative capability in the presence of high sugar concentration; fermentation rate; hydrogen sulfide production; killer activity; resistance to high ethanol and sulfur dioxide; foam production; and enzymatic profiles. Ten local wine yeast strains belonging to Saccharomyces, and one commercial active dry yeast as a reference strain were evaluated. Fermentation characteristics were evaluated in terms of kinetic parameters, including ethanol yield (Y_P/S), biomass yield (Y_X/S), theoretical ethanol yield (%), specific ethanol production rate (q_p; g/gh), specific glucose uptake rate (q_s; g/gh), and the substrate conversion (%). All tested strains were able to grow at 37 °C and to start fermentation at 30° Brix, and were resistant to high concentrations of sulfur dioxide. 60 % of the strains were weak H₂S producers, while the others produced high levels. Foam production was high, and no strains had killer activity. Six of the tested strains had the ability to grow and ferment at concentrations of 14 % ethanol. Except for one strain, all fermented most of the media sugars at a high rate, producing 11.0–12.4 % (v/v) ethanol. Although all but one strain had suitable characteristics for wine production, they possessed poor activities of glycosidase, esterase and proteinase enzymes of oenological interest. Nine of the ten local yeast strains were selected for their good oenological properties and their suitability as a wine starter culture.     

Effects of ethanol and diabetes on galactose oxidative metabolism and elimination in rats.

Blood galactose clearance after an intravenous galactose load has been widely used for years as an index of liver function. We developed a noninvasive [13C]galactose breath test, which explores galactose oxidative metabolism; this test is well correlated with liver fibrosis in patients with chronic viral hepatitis. The goal of this study was to evaluate the influence of nonhepatic factors such as diabetes and ethanol on whole-body galactose clearance (measured as the serum galactose elimination capacity test) and oxidative metabolism (measured as the [13C]galactose-induced breath 13CO2 production) in rats. Acute ethanol administration induced a significant decrease of galactose clearance and 13CO2 production. There was a significant correlation between the amount of ethanol given and the inhibition of galactose metabolism (R2 = 0.72, p < 0.0001). In streptozotocin-induced diabetic rats, the [13C]galactose-induced breath 13CO2 production was significantly reduced (p < 0.0001) and normalized by insulin treatment. However, diabetes did not decrease whole-body galactose clearance, indicating an isotopic dilution of [13C]glucose produced from [13C]galactose metabolism into the enlarged glucose pool. These results must be taken into account when using the [13C]galactose breath test as a quantitative liver function test.     

A disposable Laccase–Tyrosinase based biosensor for amperometric detection of phenolic compounds in must and wine

An amperometric biosensor for the detection of polyphenols in wine has been developed immobilizing the two enzymes Tyrosinase and Laccase on graphite screen printed electrodes modified with ferrocene. Different immobilization procedures have been carried out, the sensor operational parameters have been optimized, determining the best conditions and the analytical method for the analysis of samples. The biosensor has been then tested with real samples, using wines and musts supplied by Astra, experimental winery, in Imola (Italy). The biosensor gave good results when employed for wine analysis, showing a good agreement with the spectrophotometric data obtained with the Folin-Ciocalteu test, the official method for polyphenols’ analysis in wine. On the other hand, the measurements on musts and wines recently bottled, were seriously affected by the presence of an high level of free sulphur dioxide. SO₂ is the likely responsible for enzyme activity inhibition on the sensor. Further studies are currently proceeding to find out the most suitable conditions to obtain results not influenced by the presence of sulphur dioxide.     

Effect of low-temperature fermentation on yeast nitrogen metabolism

The aim of this study was to analyse the influence of low-temperature wine fermentation on nitrogen consumption and nitrogen regulation. Synthetic grape must was fermented at 25 and 13°C. Low-temperature decreased both the fermentation and the growth rates. Yeast cells growing at low-temperature consumed less nitrogen than at 25°C. Specifically, cells at 13°C consumed less ammonium and glutamine, and more tryptophan. Low-temperature seemed to relax the nitrogen catabolite repression (NCR) as deduced from the gene expression of ammonium and amino acid permeases (MEP2 and GAP1) and the uptake of some amino acids subjected to NCR (i.e. arginine and glutamine). Low-temperature influences the quantity and the quality of yeast nitrogen requirements. Nitrogen-deficient grape musts and low temperature are two of the main prevalent causes of sluggish fermentations and, therefore, the effects of both growth conditions on yeast metabolism are of considerable interest for wine making.     

The vinification of partially dried grapes: a comparative fermentation study of Saccharomyces cerevisiae strains under high sugar stress

AIMS: The study of the fermentation performance of Saccharomyces cerevisiae strains under high sugar stress during the vinification of partially dried grapes. METHODS AND RESULTS: Microvinification of partially dried grape must with sugar concentration of 35 degrees Brix was performed using four commercial strains to carry out alcoholic fermentation. A traditional red vinification without nutrients addition was applied. Yeasts displayed different efficiency to convert sugar in ethanol and varied in glycerol yield. Sugar consumption and ethanol level were attested at 80-87% and 143.5-158.0 g l(-1) respectively. High correlation between sugar and assimilable nitrogen consumption rate was observed. Statistical treatment of data by principal component analysis highlighted the different behaviours that strains exhibited in regard to the production of higher alcohols and other compounds important to wine quality. CONCLUSIONS: Saccharomyces cerevisiae strains displayed appreciable capability to overcome osmotic stress and to yield ethanol fermenting high sugar concentration grape must in winemaking condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provided insights on the strain contribution to wine quality subordinate to stress condition. This investigation is of applicative interest for winemaking and processing industry that use high sugar concentration musts.     

公共空间安全感研究：以上海城市街景感知为例

在文献基础上梳理了街道安全感影响因素,并采用上海样本检验了“街道眼”等西方街道安全理论。邀请30位学生和30位市民对上海5个不同发展时期社区的300张百度街景图片进行安全感评定。实验发现绿视率、管理程度、车道数等都对安全感起着显著作用,并分别建立了单双车道和多车道街道空间的安全感回归模型。其中发现绿视率（单双车道相关系数R=0.728,p<0.01;多车道相关系数R=0.471,p<0.01）、管理程度（单双车道相关系数R=0.766,p<0.01;多车道相关系数R=0.450,p<0.01）、车道数量因素（相关系数R=0.502,p<0.01）对安全感均有显著的积极作用,界面透明度（单双车道相关系数R=0.222,p<0.01）、独立自行车道（相关系数R=0.309,p<0.01）及设计美感（相关系数R=0.432,p<0.01）等因素在单双车道空间中具有积极影响,而助动车与自行车（单双车道相关系数R=-0.327,p<0.01;多车道相关系数R=-0.281,p<0.01）在对安全感知评价具有消极影响,机动车（单双车道相关系数R=0.251,p<0.01;多车道相关系数R=-0.327,p<0.01）在单双车道与多车道空间中呈现相反的作用。     

Diminution of tissue lipid peroxidation in rats is related to the in vitro antioxidant capacity of wine

Wine polyphenols could reinforce the endogenous antioxidant system, thereby diminishing oxidative damage. Studies in chronic models to understand the relationship between the bioavailability of polyphenols and their biological effects are still lacking. The aim of the present study was to prove the hypothesis that the antioxidant capacity of wines in vitro is positively correlated with the antioxidant capacity of plasma and negatively correlated with tissue lipid peroxidation, after chronic wine consumption. Adult rats received: water (control group), wine having variable phenolic content, ethanol (12.5% v/v) or alcohol-free red wine, for 4 weeks. The antioxidant capacity of wines in vitro and that of plasma induced in vivo were assessed through the reduction of ferric iron (FRAP, ferric reducing ability of plasma). Lipid peroxidation (production of thiobarbituric acid reactive substances, TBARS), and the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were determined in kidney, liver and lung. The phenolic content of wines was positively correlated with their FRAP values in vitro (r=0.407, p <0.002). Also, the relationship between wine FRAP in vitro to its respective plasma value in vivo showed a positive correlation (r=0.433, p <0.005). Phenolic concentration of wine did not influence the activity of CAT, SOD and GSH-Px of the three organs studied, but it was negatively correlated with their production of TBARS (r=-0.852, -0.891 and -0.790 for kidney, liver and lung, respectively, p <0.001). The present data provide evidence that the antioxidant capacity of wine in vitro implicates a homologous effect in vivo, thus helping to modulate tissue lipid peroxidation.     

欧美杨杂交种'中嘉8'净光合速率与若干生态因子的相关分析

本文利用DPS数据处理系统,采用多元逐步回归及通径分析方法,得出欧美杨杂交种‘中嘉8’(Populusdeltoides CL.‘Zhongjia 8’)在6月和9月净光合速率与生态因子的关系。结果表明,‘中嘉8’在6月晴天净光合速率呈单峰曲线,其最主要影响因子为光合有效辐射PAR(X1),二者极显著相关,净光合速率日变化最优方程为:Y=17.8271 0.0108X1-0.2185X2;9月晴天净光合速率呈双峰曲线,影响光合速率日变化的最主要因子为空气CO2浓度Ca(X4),净光合速率日变化最优方程为:Y=5.2915-0.0030X1 0.3414X3-0.0216X4。统计分析表明,采用多元逐步回归及通径分析应用于统计光合速率与生态因子的相关关系,较二元变量的相关分析更为科学合理。     

Managing high-density commercial scale wine fermentations

AIMS: To investigate the effects of grape juice dilution and different temperature/nitrogen addition regimes on commercial-scale, high-density Shiraz and Chardonnay fermentations. METHODS AND RESULTS: Duplicated fermentations (30 hl) were conducted at two temperatures for Shiraz and for Chardonnay. Two additional tanks of Chardonnay and Shiraz were diluted. Nitrogen was added once at inoculation or in aliquots over several days. Yeast concentration and viability was determined by flow cytometry. Fermentation chemistry was monitored by Fourier transform infrared spectroscopy. Fermentations arrested in both of the undiluted, higher temperature duplicate tanks of Shiraz. Different fermentation temperature resulted in sensorially different Shiraz, but not Chardonnay, wines made from undiluted musts. The converse was observed for wines made from diluted musts. CONCLUSIONS: High-density musts can be fermented completely using reduced fermentation temperature coupled with incremental nitrogen addition. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study in duplicated, commercial-scale, high-density grape juice fermentations to address temperature, nitrogen addition, and juice dilution effects on stuck fermentation potential and wine sensory properties.     

Typing of human and bovine Staphylococcus aureus by RAPD-PCR and ribotyping-PCR

AIMS : The aim of this study was to investigate the production of ethyl carbamate (EC) precursors by Lactobacillus hilgardii in model and Douro fortified wines and to determine the relationship between these compounds and EC levels in this type of wine. METHODS AND RESULTS: Several model fortified wines and fortified wine inoculated with L. hilgardii were analysed for citrulline and EC formation. A good correlation (R > 0.9) was obtained between citrulline and potential EC (that EC which is formed during heating of sample at 80 degrees C for 48 h). CONCLUSIONS: This correlation allowed us to calculate the potential EC formed during lactic acid bacteria activity in fortified wine. SIGNIFICANCE AND IMPACT OF THE STUDY: A good correlation was obtained (R=0.92) between measured and calculated EC in spoiled fortified wines, citrulline apparently being the main EC precursor produced by Lact. hilgardii thus contributing to the potential EC in this type of wine.     

Analyses of stress resistance under laboratory conditions constitute a suitable criterion for wine yeast selection

During wine production, yeast cells are affected by several conditions that are adverse to growth (oxidative, osmotic and ethanol stress among others) and they should detect and respond to these conditions, otherwise alcoholic fermentation can be negatively affected. In this work we have analyzed the fermentative behaviour of 14 commercial and non-commercial strains in several synthetic musts. According to the data obtained these strains have been classified into three groups depending on whether or not vinification was completed (and on the amount of residual sugar remaining in the must if it was not). Moreover, we have determined the resistance of these strains to several stress situations under laboratory growth conditions. We have been able to establish a correlation between the groups based on fermentative behaviour and resistance to several stress conditions (especially oxidative and ethanol stress), by applying discriminant analysis to the data obtained in these experiments. Our results indicate a clear relationship between stress resistance and fermentative behaviour and this opens up the possibility of using this information as a criterion for the future selection of wine yeasts.     

Biological characterisation of superficial bladder cancer by bivariate cytokeratin 7/DNA analysis, flow cytometric assessment of MIB- 1, and an immunohistochemical study.

A total of 238 cases of bladder carcinoma stages Ta, Tis, T1 were submitted prospectively to multiparameter flow cytometry and immunohistochemical study in order to determine the biological aggressiveness of the tumour. DNA index (DI), S-phase fraction (SPF) obtained by bivariate cytokeratin 7/DNA analyses, and the immunohistochemical evaluation of p53 and MIB-1 were studied in relation to the traditional prognostic factors in bladder cancer (stage and grade). the variance analysis results showed that DNA aneuploidy was significantly associated with high stage (p = 0.0001), high grade (p = 0.0001), high SPF value > or = 5.5% (p = 0.0001), MIB-1 positivity > or = 31% (p = 0.0001) and high expression of p53 (staining involving > 50% of cells, p = 0.0001). Even if there was no statistical significance the hypotetraploid class (1.70 < DI < 1.89) showed poor prognostic biomarkers more frequently than the other aneuploid classes. Out of 238 cases, 101 were also submitted to flow cytometric measurement of MIB-1 (fMIB-1) to study the correlation between cell proliferation and DNA content. Data obtained from fresh, 3:1 methanol/acetone fixed samples were compared with values obtained from both cell cycle analysis methods and routine application of the MIB-1 immunostaining in histological sections. fMIB-1 values were positively correlated with SPF values (r = 0.801, p < 0.01) and S+G2M fraction (percentage of cells in S and in G2M phases) (r = 0.763, p < 0.01) but no correlation with paraffin sections was found. A fMIB-1 value > 7% was strongly associated with aneuploidy (p = 0.0001). The determination of DNA content coupled with the study of the epithelial (cytokeratin 7) and proliferative (MIB-1) markers could be useful in providing important information on the biological behaviour of superficial bladder tumours.     

A quick screening method to identify β-glucosidase activity in native wine yeast strains: application of Esculin Glycerol Agar (EGA) medium

One hundred and fifty-four yeast strains were isolated from grapes and musts of Uruguayan vineyards and wineries. Only thirty strains showed β-glucosidase activity in Esculin Glycerol Agar (EGA) solid medium. Twenty-one were non-Saccharomyces and nine were Saccharomyces cerevisiae strains. The objective of this study was to evaluate the suitability of Esculin Glycerol Agar (EGA) solid medium for screening β-glucosidase activity in native yeasts strains. Halo sizes measured in the EGA solid medium were correlated to the Glycosyl-Glucose (GG) indexes measured after fermentation of grape musts with each strain. The two S. cerevisiae strains with the best performance were selected for further fermentations on a Muscat Miel grape must, rich in bound monoterpenes. The levels of free linalool, hodiol I and geraniol increased significantly as compared to fermentation with a commercial wine yeast strain. These results show the suitability of this simple and economic medium to identify S. cerevisiae glucosidase producers with a potential impact on real winemaking conditions. On the other hand, great variability was found for the non-Saccharomyces strains, and this would demand further studies for each species. In conclusion, the use of EGA solid medium shows that the screening method is suitable for exploring the glucosidase activity of native strains of S. cerevisiae and shows good correlation with its real impact on free aroma compounds in the final wine.     

Acceleration of cell growth and ester formation by ultrasonic wave irradiation

The effects of ultrasonic wave irradiation on cell growth and the formation of ethanol and other volatile components in the fermentation process were investigated. The fermentation periods were reduced to 50–64% in wine, beer, and sake made from saccharified rice solution when weak ultrasonic waves were irradiated at 30 mW/cm²; the total intensity was 590 mW. YEPD medium fermented by a combination of optimal thermal trajectory control and continuous ultrasonic irradiation yielded a concentration of isoamylacetate about 2.5 times greater than the maximum concentration under isothermal conditions (20°C). Irradiation had virtually no effect when the dissolved carbon dioxide concentration (DCO₂) was maintained at a level lower than the experimental value of Bunsen's CO₂ absorption coefficient. Therefore, it is suggested that irradiation accelerates the formation of ethanol and other components mainly by decreasing DCO₂.     

Changes in the activity of the general amino acid permease from Saccharomyces cerevisiae var. ellipsoideus during fermentation

The evolution of the activity of the general amino acid permease and ethanol and glucose concentrations in the medium were studied in a mild fermentation process carried out by a wine strain of Saccharomyces cerevisiae var. ellipsoideus isolated from grape musts in spontaneous fermentation. The cells displayed a reduction in the activity of the general amino acid permease parallel to the increase of ethanol in the medium. This ethanol increase was not enough to promote a substantial inhibition on the total polypeptide synthesis measured as polyuridylic-acid-directed polyphenylalanine synthesis.